The overall objective of this application is to show that 2'O-(N,N-dimethylthiocarbamate) (DMTC) protected monomers (see Fig. 1) can be used as effectively in the SPS of oligo-RNAs as the commercial ones and that an siRNA will be as biologically active as one prepared from other monomers. The DMTC group is sterically small, achiral, dipolar, and spectrally simple. It is stable to conditions throughout oligo-RNA synthesis described above. It is orthogonal to all other PGs and is readily and completely removed by aqueous oxidants. It is commercially attractive since it avoids issues of other 2'O-PGs, it is inexpensive and allows for a greener and more inexpensive process with it aqueous deprotection. The revised specific aims are based on additional preliminary studies that have demonstrated: (a) efficient synthesis of all four 2'-DMTC protected ribophosphoramidite building blocks at 200+ mg scales with improved purity; (b) high yield couplings (>99%) in the synthesis of several oligo-RNAs and (c) high yield deprotection (>XX%) of these oligos using NaIO4. Accomplishing the specific aims will show feasibility from monomer synthesis to biological activity of an siRNA. First, at least 3-5 g of pure 2'-DMTC, 5'-DMTr, acyl-protected base, cyanoethylphosphoramidites (CEP) of uridine (rU), adenosine (rA), cytidine (rC) and guanosine (rG) monomers will be prepared in scaleable batches of 1-5 grams. Second, industry standard analytical methods will be developed to confirm the identity and purity of each monomer and its final intermediate. Reference standards will be made and batches analyzed by TLC, HPLC, 1H and 31P NMR spectroscopies (>98%). Purity will be further confirmed by solubility (a clear, particulate-free 0.1M solution), loss on drying (<2%), and 3'O-DMTC levels (<0.1%). Finally, the preparation and purification of oligo-RNAs from 2'-O-DMTC monomers will be optimized in terms of reagents and exposure times as well as deprotection of all PGs, especially DMTC ones. While at least 12 batches of oligos will be prepared, the results will be used to prepare a specific siRNA that will be compared in chemical and biologically to one prepared from 2'O-TOM monomers. In Phase II the preparation of and purification of 2'O-DMTC protected monomers will be scaled-up and refined to give acceptably pure batches at near commercial quantities (>100 g) in Phase II. The automated SPS will be further optimized so that robust protocols during commercialization. Further, the compatibility of these DMTC monomers will be evaluated in the preparation of oligo-RNAs with phosphothioate, 2'OMe, 2'O-silyl protected monomers and others useful for the preparation of siRNA's and other RNAi's. Compatibility with phosphoramidites to introduce biotin, cholesterol, etc. is expected but will be verified. [unreadable] [unreadable] The preparations of oligo-RNAs for research and therapeutic purposes lag behind those of oligo-DNAs in large part because of the lower efficiency of their preparation. This in turn is largely due to the necessary 2'O-protecting group which inhibits the coupling at the nearby 3' center. Here, a novel 2'O-protecting group is described which shows very high coupling efficiencies and meets all the necessary requirements of such protecting groups. [unreadable] [unreadable] [unreadable]